The goal of this grant is to develop CMV-vectored SIV and eventually HIV vaccines that are effective in newborn infants. First-generation RhCMV-vectored SIV vaccines (RhCMV/SIV) are effective in adult animals previously infected with wild-type RhCMV but do not protect newborn animals. Possible explanations for this failure include (i) dramatically different function of the developing immune system of newborns and (ii) absence of wild-type RhCMV and its transformative effects on immunity (>98% of newborn babies are CMV uninfected). We demonstrated that a second-generation vaccine lacking the viral IL-10 gene (RhCMVdIL10/SIVgag) does protect RhCMV-seronegative older infants (8-10 months). Protection via RhCMVdIL10/SIVgag is associated with impressive transcriptomic changes similar to those reported for adult animals receiving RhCMV/SIV. Viral IL-10 function is therefore an impediment to RhCMV/SIV efficacy, particularly in younger animals that may have relatively greater function of tolerogenic, anti-inflammatory pathways. Given our favorable results in older infants, it is likely that RhCMVdIL10/SIVgag is capable of priming similar responses in newborns. To counter the possibility that newborn recipients may mount quantitatively lower T- cell responses, we additionally developed an Ad26 vector that delivers SIVgag together with dominant- negative IL-10 receptor. Delivery of antigen with dominant-negative receptor ensures that the relevant APCs, i.e., those presenting antigen, are unaffected by ambient IL-10. This unique vaccine will be used as a boost. We hypothesize that restrained IL-10 signaling in newborns vaccinated with RhCMVdIL10/SIV permits development of high-frequency, fully functional T cells that are protective against SIV. If the hypothesis is correct then this work will dramatically advance research into vaccination of newborns against HIV and other diseases, simultaneously providing measures of the needed responses and new tools to stimulate them. Aim 1: Test protective efficacy of viral IL-10-deficient RhCMV/SIV vaccines in newborn animals while comprehensively profiling innate immune subsets. Our hypothesis predicts that the viral IL-10-deficient vaccine, RhCMVdIL10/SIVgag, stimulates improved T-cell responses and significant protection in newborn animals; furthermore, protection is associated with transcriptional changes concentrated in innate cells and associated with improved T-cell function. Aim 2: Determine if newborn immune responses to RhCMV/SIV can be boosted by Ad26-vectored vaccines delivering vaccine antigen with or without dominant-negative IL-10 receptor. Adenovectors stimulate strong responses acutely and have been shown to boost protective responses to CMV-vectored vaccines. Here we test an Ad26-based booster vaccine that additionally inhibits IL-10 signaling in APCs to evade regulatory responses that are particularly strong in newborns.